Developing a bioink for single-step deposition and maturation of human epidermis

Abstract

Patients with severe burns, which cause extensive damage to their skin, require rapid intervention to prevent life-threatening hypothermia, infection, and fluid loss. Current treatments typically involve surgical excision of the burned skin and reconstruction of the wound with the aid of skin autografts. However, there is a lack of donor site in the most severe cases. While alternative treatments such as cultured epithelial autografts and “spray-on” skin can allow much smaller donor tissues to be used (and hence reduce donor site morbidity), they present their own challenges in terms of fragility of the tissues and control of the cell deposition, respectively. Recent advances in bioprinting technology have led researchers to explore its use to fabricate skin grafts, which depend on several factors, including appropriate bioinks, cell types, and printability. In this work, we describe a collagen-based bioink that allows the deposition of a contiguous layer of the keratinocytes directly onto the wound. Special attention was given to the intended clinical workflow. For example, since media changes are not feasible once the bioink is deposited onto the patient, we first developed a media formulation designed to permit a single deposition step and promote self-organization of the cells into the epidermis. Using a collagenbased dermal template populated with dermal fibroblasts, we demonstrated by immunofluorescence staining that the resulting epidermis recapitulates the features of natural skin in expressing p63 (stem cell marker), Ki67 and keratin 14 (proliferation markers), filaggrin and keratin 10 (keratinocyte differentiation and barrier function markers), and collagen type IV (basement membrane protein involved in adherence of the epidermis to the dermis). While further tests are still required to verify its utility as a burn treatment, based on the results we have achieved thus far, we believe that our current protocol can already produce donor-specific model for testing purposes.